Spray dried vaccine preparation comprising aluminium adsorbed immunogens

ABSTRACT

Vaccine preparations in stable particulate form are disclosed. An immediate-release preparation comprises an immunogen adsorbed to an aluminum adjuvant. A controlled- or delayed-release preparation comprises microspherical particles comprising a continuous matrix of biodegradable polymer containing discrete, immunogen-containing regions.

This application is a continuation of Ser. No. 08/002,485, filed Jan. 8,1993 (abandoned), and a 371 of PCT/AU93/00677 filed Dec. 24, 1993.

FIELD OF THE INVENTION

This invention relates to vaccine preparations, and in one particularembodiment it relates to vaccine preparations of the type which arevariously described as controlled- or delayed-release vaccines,pulsatile or pulsed-release vaccines and single-shot vaccines. Thepreparations of the present invention are relevant for use as human andveterinary vaccines, and are provided in the form of a dry powder, whichcan be subsequently incorporated into a liquid suspension or in a solidpellet or implant for administration. Typically, administration of thevaccine preparation of the present invention in the form of a liquidsuspension is by parenteral administration, for example by subcutaneousor intramuscular injection.

BACKGROUND TO THE INVENTION

Delivery of a full course of vaccine in a single dose has heldattraction in both human and veterinary medicine and a number of patentsand other publications (e.g. U.K. Patent No. 1,567,503) have addressedthis possibility. For veterinary applications, the advantages include:

(i) reduced time--animals need be handled only once,

(ii) reduced cost--single veterinary visit and reduced handling costs,

(iii) guaranteed compliance with recommended dose schedule (number ofdoses, time interval between doses).

In human medicine, the above three advantages are also important withcompliance being extremely important in developing countries whererepeated access to infants is often not possible. In addition, the painand suffering associated with vaccination, especially of infants, is anadditional reason to favour a single-dose vaccine in human medicine.

Early studies of vaccination using inactivated vaccines (generallytetanus or diphtheria toxoids), have demonstrated the importance of twoor more discrete doses of vaccine with an interval of at least 4 weeks,and preferably longer, between doses. A third dose is sometimesnecessary to induce an adequate immune response, especially in younganimals or infants where transfer of maternal antibodies could interferewith the preliminary immune response.

Recent studies in theoretical immunology have supported these findingsand introduced the phrase "affinity maturation". Affinity maturationdescribes the process whereby plasma cells secreting high affinityantibodies to the desired immunogen are preferentially selected whilstplasma cells secreting antibody of lower affinity are lost. The processinvolves competition between follicular dendritic cells and plasma cellsfor antigen binding and thus can only occur effectively in the presenceof limiting amounts of antigen. The process of affinity maturation maynot commence until 2 to 3 weeks after a primary vaccine dose and it isimportant that the second dose of antigen not be given until the processis effectively complete. This is readily achievable in a multidosevaccination schedule provided the first dose does not contain too muchantigen. However, for this process to be achieved in a single dosedelayed-release vaccine, it is important that the second and subsequentdoses do not release their antigen payload prematurely. To achieve this,the antigen must be contained within a matrix which has a defined timeof degradation. This matrix should be biodegradable, althoughbiocompatible matrixes have been proposed as acceptable. A number ofoptions have been reviewed by Cox & Coulter, 1992.

The major effort to develop delayed release vaccines has centred roundthe studies of Eldridge et al., 1990; 1991, who used the biodegradablecopolymer--polylactide coglycolide to produce antigen-containingmicrospheres and observed a delayed-release of the antigen contents invivo (see also Australian Patent Specifications Nos. 79929/87 and33433/89). Similar observations have been reported by Kreuter, 1990using nanoparticles produced from acrylate polymers. Although the aboveworkers were able to show that the concept of delayed-release vaccineswas possible, the process they used in the preparation of the vaccinessuffered from a number of deficiencies making it unsuitable for theroutine manufacture of a vaccine. The major problems were:

(i) exposure of biological materials to denaturing chemical and physicalconditions, and

(ii) difficulty of scale-up,

(iii) low efficiency of incorporation of hydrophilic compounds (e.g.proteins).

In European patent publication No. 0486959, in the name of VectorpharmaInternational SpA, there are disclosed controlled release, particulatepharmaceutical compositions containing pharmacologically activesubstances, the compositions comprising a biodegradable polymer such aspolylactic acid, polyglycolic acid and copolymers thereof and/or otherpolymers including a polysaccharide gellifying and/or bioadhesivepolymer, an amphiphilic polymer, an agent modifying the interfaceproperties of the particles and the pharmacologically active substance.In the preparation of the pharmaceutical composition, the polymericsubstituents are co-solubilised with the agent modifying the interfaceproperties either in the absence of any solvent or in the minimumnecessary amount of solvent, and the pharmacologically active substancesthen dissolved or dispersed in the polymer solution prior to formationof the final particles, for example by emulsion, extrusion, spray dryingor spray-congealing techniques. As previously described, this techniquesuffers from a major disadvantage in that the pharmacologically activesubstance is directly exposed to the mixture of polymeric compoundstogether with any solvents therein, which results in the denaturing ofbiological materials used as the pharmacologically active substance.

It is a principal object of the present invention to provide a vaccinepreparation and method for the production thereof wherein theimmunogenic material is not exposed to an organic solvent or otherorganic phase when in soluble form, so as to ensure that there are noconformational changes in the immunogen, in other words to maintain thenative structure of the immunogen.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there isprovided an immediate-release vaccine preparation in stable, dryparticulate form, said particles being microspherical particles preparedby spray-drying, comprising an inmmunogen adsorbed to an aluminium saltadjuvant.

In accordance with this aspect of the invention, there is also provideda method for the production of an immediate-release vaccine preparationin stable, dry particulate form as described above, which comprises thesteps of forming an aqueous suspension of aluminium salt-adsorbedimmunogen, and subsequently spray-drying said suspension.

Freeze-drying or lyophilisation of similar preparations has beendescribed by Csizer et al. (U.S. Pat. No. 4,578,270). This process has anumber of shortcomings, most importantly the need to add large amountsof both dextran and protein so that partial retention of the aluminiumgel structure can be achieved (40 and 6-4 mg/ml respectively). Thislarge addition of protein can act to displace vaccine antigens from thealuminium gel and in addition would, in most cases, be immunogenic andas a result tend to swamp the immune response to the vaccine antigen.Other problems associated with lyophilisation are that it is lessamenable to large-scale production, equipment costs are significantlyhigher and the resultant product tends to form flakes rather thanfree-flowing microgranules.

Surprisingly, the gel-forming nature of aluminium gels is completelyretained during spray-drying even in the absence of any other materials(apart from minimal quantities of vaccine antigen, typically 1 to 10μg/ml) which could exert a stabilising effect. Addition of water to thespray-dried powder results in the instant formation of a typical gel,with sedimentation properties similar to the starting material.

In accordance with a second aspect of the present invention, there isprovided a controlled or delayed-release vaccine preparation in stable,dry particulate form, said particles being microspherical particlesprepared by spray-drying comprising a continuous matrix of biodegradablepolymer containing one or more discrete, immunogen-containing regions.

In this aspect, the invention also provides a method for the productionof a controlled- or delayed-release vaccine preparation in stable, dryparticulate form as described above, which comprises the steps offorming an emulsion of an aqueous suspension comprising the imnunogenand optionally an adjuvant in a continuous organic phase having saidbiodegradable polymer dissolved therein, and subsequently spray-dryingthe water-in-oil emulsion to form said microspherical particles whichcomprise a continuous matrix of polymer containing discrete,immunogen-containing regions.

In an alternative method, these microspherical particles are produced byspray-drying a suspension of a particulate immunogen-containingmaterial, preferably an immediate-release vaccine preparation in stableparticulate form as broadly described above, and optionally an adjuvantin a continuous organic phase having said biodegradable polymerdissolved therein, to form said microspherical particles comprising acontinuous matrix of polymer containing discrete, immunogen-containingregions.

These two processes confer major advantages over methods describedpreviously, e.g. Eldridge et al. 1991, O'Hagan et al. 1991, Singh et al.1991 and Bodmeier & Cheng 1988. In the processes of Eldridge et al. 1991and Bodmeier & Chen 1988, proteins are directly exposed to the organicsolvents required to dissolve the PLG. As a result, antigens aredenatured and, because most antigens are water-soluble, poorefficiencies of incorporation result. O'Hagan et al. 1991 and Singh etal. 1991 devised complex processes to try to overcome thesedeficiencies. Neither process was amenable to commercial scale, and inaddition the former showed poor efficiency of incorporation whilst thelatter necessitated injection of large quantities of foreign proteins.

Finally, none of these methods is inherently suited to the simultaneousincorporation of adjuvant.

Both the intermediate-release vaccine preparation of this invention andthe controlled- or delayed release vaccine preparation are in the formof microspherical particles, preferably in the range of 10 nm to 250 μm,more preferably in the range of 1 μm to 100 μm.

The vaccine preparations in stable particulate form may be made up intovaccine compositions for administration by combining at least oneimmediate-release vaccine preparation and/or at least one controlled- ordelayed-release vaccine preparation with a carrier or diluent acceptablefor pharmaceutical or veterinary use. Suitable carriers or diluents foruse in the preparation of vaccine compositions for parenteraladministration are well known in the art. Alternatively, the vaccinecomposition may be produced in the form of a solid pellet or implantwith known carrier materials.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, immunogen-containingmicrospheres of the controlled- or delayed-release vaccine preparationare produced by a one-step process of manufacture with the potential fora very high throughput. The end-product is a free-flowing powder. As anormal though not essential component of the process, adjuvant isincorporated into these microspheres in association with the immunogen,and this confers a number of advantages:

(i) the immunogen is held in a selected configuration during the dryingprocess,

(ii) adjuvant is available to stimulate the immune system at everypulsed release,

(iii) during in vivo residence time, whilst delayed-release polymer isundergoing biodegradation, the immunogen is protected from thermal andperhaps enzymic denaturation by attachment to a solid support.

In work leading to the present invention, it has surprisingly been foundthat an immediate release composition can be provided in stable, soliddry form since it has been generally believed that aluminiumsalt-adsorbed immunogens could not be prepared in powder or other dryform without recourse to complex technology and excessive andunacceptable use of stabilisers (e.g. Csizer et al.). In accordance withthe first aspect of the present invention, however, it has been foundthat a stable, solid product can be produced as a free-flowing powder bydrying an alluminium salt-adsorbed immunogen produced in aqueoussuspension. The immunogen may for example be adsorbed on an aluminiumsalt adjuvant such as aluminium hydroxide or aluminium phosphate.Preferably, the suspension also contains a protein stabiliser, andsuitable stabilisers include, for example, sugars and sugar derivativessuch as trehalose, lactose, dextrose and glucosamine. The resultantsuspension is then dried, preferably spray-dried, to form a free flowingpowder. As previously described it has been found that drying of such analuminium salt-adsorbed immunogen does not denature the immunogen, nordoes it degrade the aluminium salt adjuvant, and in fact results frompreliminary experiments show that the immunogenicity of the immunogenmay be enhanced in such a powder formulation.

In accordance with the second embodiment of the invention, there isprovided a process for the manufacture of controlled- or delayed-releasemicroencapsulated vaccines. This process involves the emulsification ofvaccine immunogen, preferably in association with adjuvant, all of whichcomprises the aqueous phase, into a continuous organic phase in whichthe biodegradable polymer is dissolved. This water-in-oil emulsion isthen spray-dried under suitable conditions such as to generatemicrospheres which comprise a continuous matrix of the polymersurrounding at least one, but preferably many, pockets of immunogen inassociation with adjuvant.

It will be noted that in accordance with this process, the emulsionwhich is formed prior to spray drying is a water-in-oil emulsion, incontrast to the oil-in-water emulsions which are produced in thepreparation of the delayed-release vaccine compositions of the prior artmentioned above.

In a modification of the process just described, the microspheres may beproduced by spray-drying microdroplets which comprise a suspension ofmicro-particulate immunogen in a solution of the polymer in organicsolvent, the micro-particulate antigen being in a form which does notdissolve in the polymer solution, and preferably being theimmediate-release vaccine preparation in stable particulate formdescribed herein.

The vaccine preparations of the present invention are applicable for usewith a wide variety of immunogens known in both human and veterinaryvaccines, including for example tetanus toxoid, diphtheria toxoid,pertussis extract vaccine, influenza virus, and the like.

The biodegradable polymer used in the present invention may be anypolymer substance which is capable of existing in a nonaqueous phase,which is biocompatible and which is capable of delayed breakdown invivo. Suitable polymers include, for example polyesters,polyorthoesters, polyanhydrides and cyanoacrylates, as well as variousnatural polymers including some proteins and polysaccharides.Particularly suitable polymers for use in accordance with the presentinvention include homopolymers of D-, L- and DL-polylactic acids (D-PLA;L-PLA; DL-PLA) and polyglycolic acid (PGA), and various copolymers (PLG)thereof. Preferably, in the formation of the water-in-oil emulsion, oneor more emulsifiers are used, and suitable emulsifiers include, forexample, Tween 80, Span 85 and various lecithins andlecithin-derivatives.

Suitable adjuvants for incorporation into a delayed-release vaccinepreparation in accordance with this invention include not only thealuminium salt adjuvants previously described (aluminium hydroxide oraluminium phosphate), but also other particulate and non-particulateadjuvants which are well known in the vaccine field. Suitable adjuvantsare described, by way of example, by Cox and Coulter, 1992.

Further features of the vaccine preparations of the present inventionand the processes for the preparation thereof will be apparent from thefollowing non-limiting Examples.

EXAMPLE 1 Preparation of an Immediate Release Tetanus Vaccine

Clostridium tetani was cultured in a protein-free casein hydrolysatemedium for 6 days, at which time approximately 60 Lf/ml (in vitroflocculation units) of tetanus toxin had been produced. Bacterial cellsand debris were removed by centrifugation then the toxin concentratedand washed on a 30,000 MW cut-off ultrafiltration membrane. Formaldehydeand lysine solutions were added to a final concentration of 0.3 and 0.9%w/v respectively and toxoiding was allowed to proceed for 2 weeks at 37°C. The resultant toxoid was purified by ammonium sulphate precipitation.

Tetanus toxoid was adsorbed to the aluminium salt adjuvant (aluminiumhydroxide or aluminium phosphate) by slow addition of the antigen to thesuspension of aluminium adjuvant whilst continuously stirring. Thestirring was continued overnight. The aluminium hydroxide gel wassourced as "Alhydrogel" from Superfos, Denmark. The aluminium phosphategel was prepared by back titration of a solution of aluminium chloridewith tri-sodium phosphate. When desired, stabiliser was dissolved inwater to a concentration of 50% (w/v) then added to the adsorbed tetanustoxoid to give the required final concentration as stated in Table 1.

The aqueous suspension of aluminium salt-adsorbed tetanus toxoid wasspray-dried in a Drytec Compact Laboratory Spray Dryer equipped with a40/100/120 concentric-type nozzle at an atomising pressure of 80 psi andan outlet temperature of 60° C. The resultant microspheres had a sizerange around 3 μm in diameter and were collected as a free-flowingpowder.

EXAMPLE 2 Preparation of an Immediate-Release Diphtheria Vaccine

Cornyebacterium diphtheria was cultured in a medium incorporating caseinhydrolysate modified to have a total nitrogen content of 0.2% (w/v) andcontaining 1.5% (w/v) maltose.

Seed was grown as a 24 hour surface culture in tubes then inoculatedinto 250 ml volumes in 500 ml Erlenmeyer flasks which were incubated at35° C. for 3 days on a table rotating at 200 rpm.

Toxin was clarified by filtration to remove bacteria, concentrated to 1%the original volume by ultrafiltration (50,000 MW cut-off) then washedat that volume with half the original volume of PBS. Final purificationwas on a Sephadex G-100 column, to a purity of 2200 Lf/mg proteinnitrogen. The procedure is described in detail by Cox (1975).Formaldehyde and lysine solutions were added to a final concentration of0.3% and 0.9% (w/v) respectively and toxoiding was allowed to proceedfor 4 weeks at 37° C.

Diphtheria toxoid was absorbed to the aluminium salt adjuvant asdescribed previously for tetanus toxoid, and the aqueous suspension ofaluminium salt-absorbed diphtheria toxoid was spray-dried as describedpreviously (Example 1).

EXAMPLE 3 Preparation of an Immediate-Release Botulinum C & D Vaccine

Clostridium botulinum strains C and D were grown in a cellophane-sacapparatus modified from Sterne (1958). Growth medium external to the sacwas a modified corn steep medium which was allowed to equilibrate withPBS within the dialysis sac. Seed cultures of C. botulinum wereinoculated into the PBS and incubated at 37° C. for 18 days underanaerobic conditions. The contents of the dialysis sac were thenharvested, cells removed by centrifugation and formaldehyde to a finalconcentration of 0.5% (w/v) added. Toxoiding was allowed to occur at 37°C. until complete (7-14 days) then potency was determined as describedin the British Pharmacopoeia-Veterinary (1985).

Botulinum toxoids type C and D were mixed with Quil A (Superfos) andspray-dried as described previously for tetanus toxoid (Example 1).

EXAMPLE 4 Preparation of Bordetella pertussis Derived PTDImmediate-Release Vaccines

Cultures of Bordetella pertussis were grown in shake flasks in amodified Stainer and Sholte medium (Stainer & Sholte, 1970) containing 1mg/ml 2,6 di-methylβcyclodextrin. The flasks were incubated at 37° C.with gentle agitation at 180 rpm for 42 hrs when a cell density ofaround 2.0×10¹⁰ organisms/ml was achieved.

Pertussis toxin (PTX) was purified from the culture supernatant afterclarification by filtration. PTX was bound specifically to asialofetuinby affinity chromatography essentially as described by Sekura et al.(1985), washed, then eluted with 50 mM Tris/4M urea buffer, pH 9.0.

PTX was toxoided at pH 9.6 in the presence of 2.5 mM glutaraldehyde for48 hrs at 4° C. when reaction was terminated by addition of 9 mM lysine.The method was essentially as described in Australian PatentSpecification No. 601415 (71581/87). The resultant pertussis toxoid(PTD) was adsorbed to the aluminium salt adjuvant and spray-dried asdescribed previously.

EXAMPLE 5 Preparation of Delayed-Release Tetanus Vaccine

A. Emulsion Procedure

50:50 and 85:15 copolymers of polylactide and polyglycolide (PLG) andthe homopolymer of polylactic acid (PLA) were obtained from BirminghamPolymers Ltd., Birmingham, Ala., U.S.A. The copolymers were solubilisedto 10% w/v dissolution in either chloroform or a mix of 5 parts oftrichloroethylene and 3 parts of 1,1,2-trichloroethane. For each ofthese polymer solutions, an emulsion was produced as follows:

(a) to 93 parts of polymer solution were added 1 part of soya lecithinand 6 parts of an aqueous suspension of aluminium salt-adsorbed tetanustoxoid, or

(b) to 88 parts of polymer solution were added 1 part of a 1:5 mixtureof TWEEN 80 and SPAN 85 and 11 parts of an aqueous suspension ofaluminium salt-absorbed tetanus toxoid.

The production of the aqueous suspension of aluminium salt-adsorbedtetanus toxoid is described in Example 1 above. The mixture wasvigorously agitated using either an ultrasonic probe or a high-speedblender (e.g. a Silverson blender) to produce a stable water-in-oilemulsion with a milk-like consistency and appearance. This emulsion wasspray-dried using a Drytec Compact Laboratory Spray Dryer equipped witha 40/100/120 concentric-type nozzle at an atomising pressure of 30 psiand an outlet temperature of 35° C. The resultant microspheres had asize range around 30 μm in diameter and were collected as a free-flowingpowder. Traces of remaining organic solvent were removed by vacuumevaporation. A number of preparations were made to permit considerationof the following variables:

(a) choice of polymer--50:50 PLG 85:15 PLG PLA

(b) choice of adjuvant--aluminium hydroxide--aluminium phosphate

(c) choice of stabiliser--0, 0.5 and 5.0% trehalose.

B. Suspension Procedure

Polymer solutions were prepared as described in Section A above, thenmicrospheres of particulate immediate-release aluminium salt-adsorbedtetanus toxoid, prepared as described in Example 1, were added to afinal 1% w/v suspension. The mixture was agitated sufficiently tomaintain an even suspension and spray-dried as described in Section Aabove to a particle size around 30 μm. In some experiments, tetanustoxoid, spray-dried to small microspheres but in the absence of anyaluminium salt adjuvant, was suspended similarly in polymer solution,and larger microspheres spray-dried as described.

EXAMPLE 6 Preparation of Delayed-Release Botulinum C & D Vaccines byEmulsion Procedure

50:50 and 85:15 copolymers of PLG and the homopolymer PLA weresolubilised to 10% w/v in dichloromethane. For each of these polymersolutions, a water-in-oil type emulsion was made as follows: to 88 partspolymer solution was added 1 part of a 1:5 mixture of Tween 80 and Span85 and 11 parts of an aqueous mixture of botulinum C and D toxoids andQuil A. The mixture was vigorously agitated using a high-speed blenderthen immediately spray-dried using a Drytec Compact Laboratory SprayDryer equipped with a 60/100/120 nozzle at an atomising pressure of 15psi and an inlet temperature of 65° C. The resultant microspheres had asize range of around 20 μm diameter and were collected as a free-flowingpowder. Traces of remaining organic solvent were removed by vacuumevaporation.

EXAMPLE 7 Incorporation of Microspheres into Implants

The microspherical particles of the present invention may be formed intoimplants, particularly for implantation into subcutaneous tissues oflivestock and companion animals. This method has the advantage ofdelivering a large number of microspheres in a simple, easily packageddevice. The implants may contain "immediate" release microspheres,"delayed release microspheres" or a defined mixture of both delayed andimmediate release microspheres to give the desired release for aparticular vaccine or active immunogen. These implants are usuallycylindrical in shape and produced by standard pharmaceutical tablettingprocedures. Various excipients may be added to aid in the compressionand tabletting processes such as calcium phosphate, Emcompress®,lactose, dextrose, lysine and magnesium stearate. Other excipients suchas a disintegrant (e.g. sodium starch glycolate; Explotab®) may also beadded to increase the dispersion characteristics of the microspheresupon implantation. The size of the implants may be varied depending onthe amount of microspheres required per dose, but would normally be inthe range of 2-4 mm in diameter and 3-10 mm long. Further polymercoatings may be applied to the implants to accelerate or retard therelease of the active immunogen following implantation.

EXAMPLE 8

A. Testing of Vaccine Preparations

(a) In vitro testing

Aluminium phosphate gel was solubilised by dilution to 2 mg AlPO₄ /ml insaline containing a final concentration of 10% w/v sodium citrate.Samples were incubated at 37° C. overnight or until completely clear.This treatment yielded an aqueous solution in which previously boundprotein molecules were freed for assay as described below.

Tetanus prototoxin

Purified tetanus prototoxin was produced by exaction from cells ofClostridium tetani harvested prior to commencement of autolysis.Preferably, cells were harvested 72 to 90 hr after inoculation andimmediately chilled at 4° C. and held at that temperature duringsubsequent processing. The culture was centrifuged at 10,000 g for 25min, washed twice in 0.15M NaCl then resuspended to 1/30 the originalvolume in 1M NaCl, 0.1M sodium citrate pH 7.5 containing 1 mMphenylmethysulphonyl chloride (PMSF), 1 mg/ml pepstatin and 1 mg/mlleupeptin. After 16 h, extracted prototoxin was separated from celldebris by centrifugation. Initial prototoxin purification was byprecipitation and washing with 40% saturated ammonium sulphate, followedby resuspension in 0.1M phosphate buffer pH6.8 containing 1 mM PMSF and1 mg/ml each of pepstatin and leupeptin. Final purification wasperformed in this buffer on an anion exchange column of an FPLC.

Diphtheria toxin

Purified diphtheria toxin as described in Example 2 with a purity of2200 Lf/mg protein nitrogen.

Botulinum C and D toxin

Botulinum toxins were clarified by filtration then concentrated andpartially purified by ultrafiltration (50,000 MW cut-off).

B.pertussis PTX

PTX was purified as described in Example 4. Final purity was in excessof 99%.

Enzyme immunoassay

Purified antigen was diluted to 10 μg/ml (approx 5 Lf/ml) in 0.05Msodium bicarbonate buffer pH 9.6 and used to coat polystyrene plates(Maxisorb/NUNC, Denmark) overnight at 4° C. At the end of thisincubation, contents were aspirated and the wells post-coated withcasein solution (1 mg/ml) in 0.01M phosphate buffered saline (PBS) pH7.2 for 1 hr at 20° C. Contents were again discarded, the wells wererinsed with stabilising solution then plates were dried and stored insealed metal foil pouches.

All serum dilutions were performed in Blue Diluent (CSL, Australia) aPBS Tween diluent containing casein. Test samples were incubated for 60min at 20° C. (0.1 ml/well), washed 6 times with PBS Tween thenincubated similarly with horse radish peroxidase (HRP)-conjugated sheepanti-mouse Ig, sheep anti-horse Ig, or rabbit anti-sheep Ig. Peroxidaseactivity was measured by addition of 0.1 ml/well of substrate solutioncontaining H₂ O₂ and tetramethyl benzidine. After 5 min at roomtemperature, the reaction was stopped by addition of 0.05 ml/well 0.5MH₂ SO₄. Absorbance readings were made at 450 nm on an automated EIAplate reader. Titration of a subsidiary standard serum of 115 IU/ml wasincluded on all plates.

(b) In vivo testing

Naive mice were dosed subcutaneously with a total of 0.1 to 1.0 Lftetanus toxoid and 1 μg of each of diphtheria toxoid and PTD. Mice wereeyebled at regular intervals and their serum antibody levels tested byEIA using the above described validated assay.

Sheep were dosed with bivalent botulinum which contained 2 cpu/dosebotulinum C toxoid, 21.8 cpu/dose botulinum D toxoid and 0.4 mg Quil A.

Horses were dosed with 10 Lf/dose of tetanus toxoid by the intramuscularroute.

B. Results for immediate release preparations

Table 1 shows in vitro and in vivo testing data for a range ofmicrospherical immediate-release vaccine preparations produced inaccordance with Example 1. Group 7 was the positive control; titres of100 are considered background. It can be seen that inclusion of 5%trehalose along with tetanus toxoid adsorbed to either aluminiumhydroxide or phosphate permitted the formation of microspheres with ahigh retention of in vitro activity and no reduction in immunogenicity(Group 2 and 6).

                  TABLE 1    ______________________________________                              MEDIAN   % EIA    GROUP   DOSE              TITRE    Activity    ______________________________________    1       TT-AIPO.sub.4 s/d 100      3.9    2       TT-AIPO.sub.4 + 5% trehalose                              1500     70.2    3       TT-AIPO.sub.4 + 5% glucosamine                              1150     74.3    4       TT-AIPO.sub.4 + 5% lactose                              900      74.4    5       TT-Al(OH).sub.3 s/d                              100      N.T.    6       TT-Al(OH).sub.3 + 5% trehalose                              1550     N.T.    7       TT-AlPO.sub.4 suspension                              1540     100    8       TT-Al(OH).sub.3 suspension                              1600     N.T.    9       TT s/d            100      67.0    ______________________________________

Table 2 shows in vivo testing data in mice for microsphericalimmediate-release diphtheria-toxoid (DT) and B. pertussis PTD vaccines.It can be seen that in all cases, the dried microparticulate vaccine wasat least as immunogenic as the liquid vaccine from which it was producedwhen given at the same dose level based on the assumption of zero lossesduring drying.

                  TABLE 2    ______________________________________               Median antibody titre (week)    Group  Vaccine   2       4    8    12   16    20    ______________________________________           DT-AIPO.sub.4    1      dried     1600    850  700  530  500   200    2      liquid    500     NT   500  240  190   130           PTD-AIPO.sub.4    3      dried     150     430  190  45   60    40    4      liquid    40      190  NT   NT   52    36    ______________________________________     NT = not tested

Table 3 shows in vivo testing data in horses for microsphericalimmediate release tetanus vaccine. Results show secondary titres 4 weeksafter the second dose (doses were given at 4 wk intervals). All horseswere seronegative 4 weeks after primary immunisation. It can be seenthat horses that were boosted with the dried vaccine (horses 1, 2 and 7)have titres on average which are better than the horse that received theliquid vaccine boost (horse 8).

                  TABLE 3    ______________________________________                        Titre    Number   Dose (secondary) 4 week  8 week    ______________________________________    1        Dried immediate release                              ND      500    2        "                ND      7500    3        85:15 delayed release*                              ND      100    4        "                ND      500    5        "                ND      400    6        "                ND      400    7        Dried immediate release                              ND      12500    8        Liquid vaccine   ND      1000    9        none             ND      ND    10       none             ND      ND    ______________________________________     ND = not detectable.     * Delayedrelease component given in combination with primary dose.

Table 4 shows in vivo testing data in sheep for microspherical immediaterelease botulinum C & D vaccine adjuvanted with Quil A. Five animalswere dosed per group. It can be seen that 2 doses of driedimmediate-release vaccine has given identical results to 2 doses ofnormal liquid vaccine over a 12 week examination period (group 2 ofgroup 1).

                  TABLE 4    ______________________________________                             Antibody    Dose                     titre (units)    Number          1°     2°    Wk 4 8    12    ______________________________________    1     liquid        liquid       1.3  14.3 3.4    2     dry immed. release                        dry immed. release                                     2.0  12.0 4.4    3     50:50 delay release                        "            12.2 3.9  1.3    4     85:15 delay release                        "            5.8  2.1  3.6    5     dry immed. release +                        "            7.2  2.3  1.0          50:50 delay release    6     dry immed. release +                        "            6.9  8.3  1.7          85:15 delay release    ______________________________________

C. Results for delayed-release preparations

Table 5 shows the in vivo testing for a range of microspherical,delayed-release vaccines. All mice were dosed with 1 Lf tetanus toxoidexcept group 1 which are the negative controls. Group 2 is the positivecontrol (liquid aluminium adsorbed tetanus toxoid) and group 3 isunadsorbed antigen. Groups 4 to 7 were prepared by the suspensionprocedure, groups 8 to 13 by the emulsion procedure. Substantial delayedrelease responses can be seen, especially for groups 9, 11 and 13.

                  TABLE 5    ______________________________________    Group                  MEDIAN Ab TITRE    No.    VACCINE         2 Wks    4 Wks 8 Wks    ______________________________________    1      no treatment    100      100   100    2      TT-AlPhos susp  2250     4200  5950    3      soluble TT      350      300   400    4      50:50 PLG s/d TT                           850      700   2500    5      50:50 PLG s/d TT-AlPhos                           700      500   3950    6      85:15 PLG s/d TT                           700      1350  1700    7      85:15 PLG s/d TT AlPhos                           1450     1050  2700    8      50:50 PLG liq TT                           900      1000  2950    9      50:50 PLG liq TT-AlPhos                           700      1450  6300    10     85:15 PLG liq TT                           550      550   1750    11     85:15 PLG liq TT-AlPhos                           500      700   6900    12     100% PLA liq TT 550      450   4300    13     100% PLA liq TT-AlPhos                           600      450   5350    ______________________________________     s/d = spraydried     TT = tetanus toxoid

Table 6 shows the in vivo testing for a range of microsphericaldelayed-release vaccines. These vaccines were prepared using trehalose(5% w/v) as antigen stabiliser and mice were dosed with 0.1 Lf tetanustoxoid per dose. All vaccines were given as a single dose and bleedstaken for assay at the specified time. The results show significantdelayed release responses for all the delayed-release vaccines ascompared with an erosion in response for groups which receivedimmediate-release vaccine only, either liquid or dried (groups 1 to 3).

                  TABLE 6    ______________________________________    GP                      Median EIA Titre    No.  Vaccine      Nozzle    2 wks                                     4 wks                                          8 wks                                               12 wks    ______________________________________    1    TT-AIPO.sub.4          100  100  100  100         suspension    2    TT-AIPO.sub.4 spray                      40/100/120                                200  200  300  200         dried    3    TT-AIPO.sub.4 spray                      60/100/120                                250  200  150  100         dried    4    no treatment           100  100  100  100    5    TT-AIPO.sub.4 in 50:50                      40/100/120                                750  1200 6750 1100         PLG    6    TT-AIPO.sub.4 in 50:50                      60/100/120                                500  500  4650 450         PLG    7    TT-AIPO.sub.4 in 85:15                      40/100/120                                500  700  2200 1650         PLG    8    TT-AIPO.sub.4 in 85:15                      60/100/120                                550  600  2250 2400         PLG    9    TT-AIPO.sub.4 in 100%                      40/100/120                                500  800  900  600         PLA    10   TT-AIPO.sub.4 in 100%                      60/100/120                                350  800  1700 2100         PLA    ______________________________________     Note:     all preparations contain 5% trehalose.

Table 3 shows the results of tetanus toxoid vaccines in horses. It canbe seen that a single dose of immediate-release vaccine (horses 9 and10) failed to induce detectable antibody levels in horses over the 8week study period. Conversely, horses which received a single dose ofvaccine which contained both immediate and delayed-release components(horses 3 to 6) showed definite antibody titres at week 8 as a result ofthe delayed release. Titers at 8 weeks were not as high as for horsesreceiving 2 doses of vaccine, but it is expected that titres willpersist longer.

Table 4 shows the results of botulinum C and D vaccination of sheep. Itcan be seen that the single dose delayed-release vaccines both on theirown (groups 3 and 4) and in combination with immediate release vaccinehave given titers at 12 weeks comparable to two doses ofimmediate-release vaccine and well in excess of that expected from asingle dose of immediate release vaccine. It is of further significancethat this result was obtained using Quil A as the adjuvant.

EXAMPLE 9 Preparation of Controlled-Release Vaccine by the SuspensionRoute Using Spray-Drying

This method was used with small samples of toxoid suspensions, typically0.6 to 1 gm in 10 ml or 1.6 to 2 gm in 60 ml, which could not beprocessed in a standard spray dryer. A special spray dryer able toprocess such small quantities was set up which atomised the slurry usinga piezoelectrically powered ultrasonic nozzle, (Sono-Tek Corp. Model8700-120MS). The advantage of this nozzle is that it is able to createsmall droplets without the use of large volumes of a second fluid or theuse of high pressure which requires much space and surface making asmall amount of material difficult to collect. The unit was providedwith a source of hot air, cold air and a filter. The hot air carried theatomised spray allowing the water to evaporate. Cold air was then mixedwith the hot air before collecting the dried sample on a filter. Thebottom of the 11 cm diameter filter was connected to a source of vacuum.There were 3 thermocouples in the device, one at the top near thenozzle, one in the middle of the device and one at the filter.

Two types of samples were processed, (1) a "saturated" preparation of30,000 Lf tetanus toxoid adsorbed on AlPO₄, each bottle containing 0.6 gAlPO₄ in 12 ml of pyrogen free water and (2) a "saturated/3" preparationof 30,000 Lf tetanus toxoid adsorbed on 1.65 g AlPO₄ in 60 ml of pyrogenfree water. Both samples were sterile. Feed rate of the slurry which wasadded under agitation conditions was between 1 and 3 ml/min. The powerto the nozzle which operated at 120 khz was about 7 watt. The filter wasset to 6 in mercury of vacuum and the cold air at 500 cm³ /min. Typicalthermocouple readings were 63 to 86° C. at the top near the nozzle, 83to 85° C. in the middle and 46 to 58° C. at the bottom near thecollection filter.

Micrographs of the product showed 5 to 20 micron spheres for the moredilute samples and 10 to 30 micron for the more concentrated samples.Solid recovery ranged from 40 to 70%.

The product from the "saturated" samples was divided into 3 portionseach being 0.865 g solids. Three solutions were prepared each 20 gmpolymer in 200 ml of solvent. Poly(DL-lactide) (inherent viscosity 0.73at 0.5 g/dl) and 85/15 Poly(DL-lactide-co-glycolide) (inherent viscosity0.65 at 0.5 g/dl) were dissolved in trichloroethylene 5 parts, 3 parts1,1,2-trichloroethane, whereas 50/50 Poly(DL-lactide-co-glycolide)(inherent viscosity 0.71 at 0.5 g/dl) was dissolved in chloroform 5parts, 3 parts 1,1,2-trichloroethane. The solids were dispersed in thesolution and stirred for 30 minutes before being sprayed with atwo-fluid nozzle (Spraying Systems, fluid cap 40100, air cap 120) at 20psi. It was sprayed into room temperature air and after drying recoveredon cloth filters. The particles were separated on a 104 micron sieve.

The product from the "saturated/3" samples was treated essentially thesame way, except in this case 1.5 gm sample was available for each finalspray run, therefore the ratio of toxoid solids to polymer was 1.5 to 20in this case instead of 0.965 to 20 for the "saturated" case.

EXAMPLE 10 Preparation of Polylactide Encapsulated Toxoid by theSpray-Dried Emulsion Technique

20 g of Poly(DL-lactide) supplied by Birmingham Polymers, Inc. (inherentviscosity 0.73 g/dl) was dissolved in a mixture of 105 ml oftrichloroethylene and 70 ml toluene by stirring overnight with amagnetic spin bar. Sodium dioctyl sulfosuccinate also known as sodiumdocusate (2.0 gm) was added with stirring until dissolution wascomplete. The saturated aqueous suspension of aluminium salt-adsorbedtetanus toxoid (12 ml) was added in a thin stream to the polymersolution which was stirred with a spin bar. Also, an ultrasonic probewas included to provide the necessary shear for stable emulsionpreparation with this small volume of solution. The mixture was fed bysyringe to a 2-fluid spray nozzle contained in a spray drying chamber atapproximately 30 ml/min. The inlet temperature to the spray chamber wasat ambient and nozzle pressure was controlled at 20 psi. The productafter drying was removed from the outlet filter socks.

REFERENCES

1. Cox, J. C. and Coulter, A. R. (1992), "Advances in adjuvanttechnology and application" in Animal Parasite Control UtilisingBiotechnology, ed. W. K. Yong, CRC Press Inc., Boca Raton, Fla.,pp49-112.

2. Eldridge, J. H., Hammond, C. J., Meulbroek, J. A., Staas, J. K.,Gilley, R. M. and Tice, T. R. Controlled vaccine release in thegut-associated lymphoid tissues. I. Orally administered biodegradablemicrospheres target the Peyer's patches, J.Control. Release, 11, 205,1990.

3. Eldridge, J. H., Staas, J. K., Meulbroek, J. A., McGhee, J. R., Tice,T. R. and Gilley, R. M. Biodegradable microspheres as a vaccine deliverysystem. Mol. Immunol. 28, 287, 1991.

4. Kreuter, J. Large-scale production problems and manufacturing ofnanoparticles, in Specialized Drug Delivery Systems. Manufacturing andProduction Technology. Tyle, P., Ed., Marcel Dekker, New York, 1990,257.

5. O'Hagan, D. T., Jeffery, H., Roberts, M. J. J., McGee, J. P. andDavis, S. S. Controlled release microparticles for vaccine development.Vaccine 9, 768, 1991.

6. Singh, M., Singh, A. and Talwar, G. P. Controlled delivery ofdiphtheria toxoid using biodegradable poly(D,L-lactide) microcapsules.Pharm. Res. 8, 958, 1991.

7. Bodmeier, R. and Cheng, H. Preparation of biodegradablepoly(±)lactide microparticles using a spray-drying technique.J.Pharm.Pharmacol, 40, 754, 1988.

8. Cox, J. New methods for the large-scale preparation of diphtheriatoxoid: Purification of toxin. App. Microbiol. 29, 464, 1975.

9. Sterne, M. The growth of Brucella abortus strain 19 in aerateddialysed medium. J. Gen. Micro. 18, 747, 1958.

10. British Pharmacopoeia-Veterinary (1985). Her Majesty's StationeryOffice, London.

11. Stainer, D. and Sholte, M. A simple chemically defined medium forthe production of Phase I Bordetella pertussis. J. Gen. Micro. 63, 211,1970.

12. Sekura, R. D., Zhang, Y. I. and Quentin-Millet, M.-J. B. Pertussistoxin: Structural elements involved in the interaction with cells.Pertussis toxin. Ed. Sekura et al., Academic Press, p.45, 1985.

We claim:
 1. A vaccine preparation in stable, dry particulate form,comprising microspherical particles prepared by spray-drying, saidparticles comprising an immunogen adsorbed to an aluminum salt adjuvant,said vaccine preparation being a free flowing powder.
 2. The vaccinepreparation of claim 1, wherein said aluminium salt adjuvant isaluminium hydroxide or aluminium phosphate.
 3. The vaccine preparationof claim 1 further comprising a protein stabiliser.
 4. The vaccinepreparation of claim 3, wherein said stabiliser is a sugar or sugarderivative.
 5. The vaccine preparation of claim 4 wherein saidstabiliser is selected from the group consisting of trehalose, lactose,dextrose and glucosamine.
 6. A method for the production of a vaccinepreparation of claim 1, which comprises the steps of forming an aqueoussuspension of aluminium salt-adsorbed immunogen, and subsequentlyspray-drying said suspension.
 7. A vaccine composition comprising atleast one vaccine preparation of claim 1, together with apharmaceutically or veterinarily acceptable carrier or diluent.
 8. Thevaccine composition of claim 7 wherein the carrier or diluent issuitable for parenteral administration.
 9. The vaccine composition ofclaim 7, wherein said carrier is a solid carrier and said vaccinecomposition is in the form of a solid pellet or implant.
 10. A method ofvaccinating a human or other animal patient, which comprisesadministration to the patient of a vaccine composition of claim
 7. 11. Avaccine preparation in stable, dry particulate form, comprisingmicrospherical particles prepared by spray-drying said particlescomprising a continuous matrix of biodegradable polymer containing oneor more discrete, immunogen-containing regions, said vaccine preparationbeing a free flowing powder.
 12. The vaccine preparation of claim 11,wherein said immunogen-containing regions also contain an adjuvant. 13.The vaccine preparation of claim 11, wherein said immunogen-containingregions contain particles comprising an immunogen adsorbed to analuminium salt adjuvant.
 14. The vaccine preparation of claim 11,wherein said biodegradable polymer is selected from the group consistingof polylactic acid, polyglycolic acid, and copolymers thereof.
 15. Amethod for the production of a vaccine preparation of claim 11, whichcomprises the steps of forming an emulsion of an aqueous suspensioncomprising immunogen and optionally an adjuvant in a continuous organicphase having biodegradable polymer dissolved therein, and subsequentlyspray-drying the water-in-oil emulsion to form microspherical particles.16. The method of claim 15, wherein said emulsion includes anemulsifier.
 17. The method of claim 15, wherein the immunogen isadsorbed to an aluminium salt adjuvant.
 18. A method for the productionof a vaccine preparation of claim 11, which comprises the steps offorming a suspension of a particulate immunogen-containing material andoptionally an adjuvant in a continuous organic phase havingbiodegradable polymer dissolved therein, and subsequently spray-dryingthe suspension to form microspherical particles.
 19. The method of claim18, wherein the particulate immunogen-containing material comprises animmunogen adsorbed to an aluminium salt adjuvant.
 20. A vaccinecomposition comprising at least one vaccine preparation of claim 11,together with a pharmaceutically or veterinarily acceptable carrier ordiluent.
 21. The vaccine composition of claim 20 further comprising atleast one vaccine preparation in stable, dry particulate form,comprising microspherical particles prepared by spray-drying, saidparticles comprising an immunogen adsorbed to an aluminum salt adjuvant,said vaccine preparation being a free flowing powder.
 22. The vaccinecomposition of claim 21, wherein said vaccine preparation comprises animmunogen adsorbed to an aluminium salt adjuvant.
 23. The vaccinecomposition of claim 20, wherein the carrier or diluent is suitable forparenteral administration.
 24. The vaccine composition of claim 20,wherein said carrier is a solid carrier and said vaccine composition isin the form of a solid pellet or implant.
 25. A method of vaccinating ahuman or animal patient, which comprises administration to the patient avaccine composition of claim
 20. 26. A vaccine preparation in stable,dry particulate form comprising microspherical particles prepared byspray drying an emulsion of an aqueous suspension comprising immunogenin a continuous organic phase having biodegradable polymer dissolvedtherein or by spray drying a suspension of particulateimmunogen-containing material in a continuous organic phase havingbiogradable polymer dissolved therein, said vaccine preparation being afree flowing powder.